This proposal is focused on a highly conserved RNA degradation pathway-Nonsense-Mediated RNA Decay (NMD)-that was originally defined as a quality control mechanism that degrades aberrant mRNAs, but now is recognized as an important regulator of normal gene expression. Specific NMD branches have been defined, each of which degrades different subsets of normal mRNAs, leading to the hypothesis that each branch regulates distinct biological events. My laboratory is focused on a specific branch of the NMD pathway that depends on two related proteins-UPF3A and UPF3B-encoded by an evolutionarily ancient gene paralog pair that has existed since the emergence of vertebrates. There is considerable interest in the UPF3- dependent branch of NMD because mutations in UPF3B in humans cause intellectual disability. Furthermore, mutations in both UPF3A and UPF3B are significantly associated with neuro-developmental disorders in humans. In this application, we propose to decipher the underlying mechanisms by which NMD controls nervous system development and function by using Upf3a- and Upf3b-mutant mouse models we recently generated. We find that Upf3b-null mice have behavioral defects that mimic some of those in humans harboring UPF3B mutations. These mutant mice also have striking defects in olfactory neurogenesis that suggest that NMD is critical for olfactory sensory neuron (OSN) survival, maturation, and axon guidance. In this proposal, we leverage the technical advantages of the olfactory system and our mouse models to elucidate the underlying mechanisms by which NMD controls nervous system development and function at both the biological and molecular levels. In Aim 1, we will follow-up on our preliminary studies suggesting that UPF3B controls the balance of the two major types of OSNs, their connections with the central nervous system (CNS), and olfactory behavior. In Aim 2, we propose to use cutting-edge methods to identify-genome-wide-the mRNAs targeted for decay by UPF3B in vivo. This is important, since very few direct NMD target mRNAs have been identified, and most of those that have been identified were discovered in cell lines. Another neglected area of investigation that we will address is whether NMD differentially regulates target mRNAs in different regions of a cell. This is important since it is becoming increasingly clear that many polarized cells, including neurons, sub-compartmentalize events to increase efficiency. In Aim 3, we focus on UPF3A and its relationship with UPF3B. We previously reported that UPF3A protein is dramatically stabilized in response to loss of UPF3B, which suggests that the latter compensates for the former, a postulate that is supported by clinical evidence. We recently obtained preliminary evidence that UPF3A can also function as a NMD repressor. By comparing both the olfactory defects and mis-regulated transcripts in Upf3a/Upf3b double-KO mice with those of Upf3a and Upf3b single-KO mice, as well as compound heterozygotes, we propose to define unique, redundant, and antagonistic functions of UPF3A and UPF3B in vivo.